Store-operated calcium entry, or SOCE, via calcium release activated calcium (CRAC) channels regulates NFAT dependent gene expression required for lymphocyte activation and generation of immune response against antigenic insults. Orai (or CRACM) proteins form the pore subunit of CRAC channels in the plasma membrane (PM), and endoplasmic reticulum (ER) resident stromal interaction proteins (Stims) act as store calcium sensors that oligomerize and translocate to ER-PM junctions to activate CRAC channels. However, the final steps involved in CRAC channel activation inside live cells have remained elusive. For instance, until recently it was believed that Stim1-Orai1 clustering in ER-PM junctions is sufficient to activate SOCE. However, in the absence of alpha-soluble NSF attachment protein (?-SNAP), a novel essential component of active CRAC channel complex that we have recently identified, Stim1-Orai1 clusters show a strong defect in activating SOCE. ?-SNAP directly binds Stim1 as well as Orai1 and regulates the ratio of Stim1:Orai1 molecules within CRAC channel clusters; a known determinant of CRAC channel activity and calcium selectivity. Furthermore, ?-SNAP deficient mouse primary CD4 T lymphocytes show a specific defect in SOCE, NFAT activation and cytokine gene expression. In Aim1, we propose to test the hypothesis that ?-SNAP facilitates a structural change within Stim1 that enables the Stim1-Orai1 clusters to acquire optimal Stim1:Orai1 ratios required for the functionality of CRAC channel clusters in live cells. To test our hypothesis, we will use a variety of cutting edge biochemical, structural and imaging approaches along with genetic tools. In Aim 2, we will use a combination of novel super resolution microscopy approaches to construct the 3 dimensional molecular architecture of CRAC channel macromolecular assembly in ER-PM junctional space of wildtype and ?-SNAP depleted cells. In summary, we will employ novel and highly interdisciplinary approaches to gain much needed insights into the molecular mechanism of SOCE activation via CRAC channels. Identification of new non-redundant components of the CRAC channel complex and detailed insights into their mechanism of action will be highly beneficial for drug design against autoimmune and allergic disorders.